RecA+ protein (wild type) is a 37,842 dalton protein found in the bacterium Escherichia coli, which is important for homologous DNA recombination. Most information about its biochemistry and enzymology comes from in vitro studies using purified RecA+ protein. Numerous in vitro studies have shown that RecA+ protein is intimately involved in the pairing reaction between homologous DNA sequences that ultimately leads to homologous recombination events (Radding; see Cox et al. or Roca et al. for recent reviews of RecA+ protein properties). It is this pairing reaction that makes RecA+ protein highly useful for DNA diagnostics applications.
In the presence of ATP, RecA+ protein catalyzes strand exchange between a number of substrates, the most relevant for DNA probe applications being single- and double-stranded DNAs. RecA protein coated single-stranded DNA probes interact with the homologous portion of a double-stranded ("native") target sequence initially by forming a recombination intermediate containing hybridized, partially joined molecules called (single) D-loops (or in some cases triple-stranded structures) (Shibata et al., 1979). This is followed by branch migration, and forming of fully hybrid molecules between the original single- and double-stranded DNAs, depending upon the extent of their homology.
Short displacement loops or triple-stranded D-loop structures in linear targets are usually unstable after deproteinization. RecA protein has been shown to form stable complexes with short oligonucleotides, between 9 and 20 bp (or larger) in length, in the presence of ATP.gamma.S and excess RecA protein (Leahy et al.). When linear double-stranded targets are used, stable probe target pairing after RecA removal appears to require (i) a homologous region of at least 38 to 56 bp, and (ii) the location of the probe target homology at the end of the linear duplex (Hsieh et al. 1990; Gonda et al.).
Rigas et al. reported that a single-stranded 43-mer could form a single D-loop complex stable to deproteinization when double-stranded negatively supercoiled circular plasmid DNA was used as the target.
When a double-stranded negatively supercoiled circular target DNA is used, RecA coated single-stranded oligonucleotide probes can also be stabilized by psoralen crosslinking before removal of the RecA protein: probe-target single D-loop products can be recovered if the oligos are at least 30-mer size (Cheng et al., 1989). To obtain psoralen crosslink stabilized single D-loop probe-target complexes when double-stranded linear DNA duplexes are used as target DNA, the probes must be at least 80 to 107-mer size (Cheng et al., 1988): these reactions are very low efficiency when compared to similar reactions with negatively supercoiled circular targets.
Experiments performed in support of the present invention have demonstrated that probe:target DNA complexes, which are stable to deproteinization, can be generated in RecA protein catalyzed reactions providing that duplex probes, which contain sequences complementary between probe strands, are used in the hybridization reactions. This discovery provides a number of opportunities for diagnostic application that exploit this stable RecA protein catalyzed probe:target hybridization complex.